This proposal is responsive to the broad Challenge Area "Enabling Technologies" and specifically is directed to 06-GM-102* Chemist/biologist collaborations facilitating tool development through collaborations between a chemist(s) and a biologist(s). Recent advances in single-cell technology have revealed cell-to-cell transcriptional differences in isogenic populations of bacteria living in the same homogeneous environment. Differences of this kind can be due to bistability, a reversible epigenetic mechanism of gene regulation that causes the appearance of two populations, one fraction that expresses a set of genes and the other in which the same genes are transcriptionally silent. Though bistability appears to be a very general and highly evolved adaptive strategy by which microbes contend with dynamic environments, there is currently no way to survey an entire genome in an unbiased manner for genes that exhibit bistability in response to a variety of environmental conditions. During this two year project, a team of chemists and biologists will develop a robust and broadly applicable method to characterize the expression behavior of each gene in a bacterial genome, by individual cells, in response to a suite of biologically relevant conditions. To achieve this, we will pursue a novel strategy that combines a primary genetic screen followed by a second screen using a microfluidic platform. We will test the functionality of this technology by determining if it can detect previously identified V. cholerae genes whose expression is under bistable control. A V. cholerae library will be created composed of cells with all possible genes fused to unstable green fluorescent protein (GFP) as a reporter. Entries of this library will be screened using a novel microfluidic platform that interrogates cells for gene expression, one at a time, with a high throughput. This approach is unprecedented and such a tool, when perfected, should contribute to an improved understanding of the ecology and control of infectious diseases. The current proposal would positively impact the economy by creating or retaining seven jobs at Stanford University and through the possible development of a new scientific industry. PUBLIC HEALTH RELEVANCE: The control of infectious diseases is impeded by the capacity of small numbers of bacteria to persist during antibiotic treatment. This persistor population is responsible for the need to use long courses of therapy and for therapeutic failures. This project will address this public health need by developing a method and tool that will identify how a fraction of a treated population of bacteria becomes drug tolerant during treatment. This understanding will lead to the development of new drugs and treatment strategies.